Enhanced osteogenic activity of bone morphogenetic protein-2 by 2-O-desulfated heparin

The objective of this study was to investigate the effect of heparin sulfate groups on the osteogenic activity of bone morphogenetic protein-2 (BMP-2) in vitro and in vivo. Three types of desulfated (DS) derivatives of heparin (2-O-DS, 6-O-DS, and N-DS) were prepared and their bioactivity in rat bone marrow derived mesenchymal stem cells (MSC) in the absence or presence of BMP-2 was evaluated. When cultured with the 2-O-DS derivative and BMP-2 MSC showed enhanced proliferation, alkaline phosphatase activity, and Runx2 mRNA expression, compared with heparin and other derivatives. A similar tendency was observed for MSC cultured on two-dimensional substrates coated with heparin or the derivatives and in three-dimensional hydrogels containing heparin or the derivatives. A binding experiment demonstrated a greater binding affinity of 2-O-DS for BMP-2 than that of heparin and the other derivatives. Following implantation into the back subcutis of mice significantly greater ectopic bone formation in terms of bone weight, amount of calcium, and histology were observed for the gelatin hydrogels incorporating 2-O-DS and containing BMP-2. In addition, the gelatin hydrogels incorporating 2-O-DS showed controlled release of BMP-2 in vitro and in vivo. These findings demonstrated that the 2-O-DS derivative of heparin has a synergistic effect on the in vitro and in vivo osteogenic activity of BMP-2.